Ingestion of Listeria monocytogenes (Lm)-contaminated food results in human disease ranging in severity from mild, self-limiting gastroenteritis to life-threatening septicemia and meningoencephalitis. The specific factors that influence disease severity are not well understood, and our knowledge of the intestinal phase of listeriosis, in particular, is severely limited. We recently developed a mouse model of foodborne infection to study the interaction of Lm with intestinal innate immune cells. In preliminary studies, we found that the majority of Lm in the gut were extracellular. This was an unexpected result, because intracellular growth and spread from cell-to-cell without encountering the extracellular milieu are generally regarded as the primary virulence strategies for these facultative intracellular bacteria. LplA1-deficient Lm that were unable to replicate intracellularly could readily invade the gut mucosa and establish infection in the underlying lamina propria, but did not persist as well as wildtype Lm, and by three days post-infection had a severe defect in spreading to the mesenteric lymph nodes (MLN). This suggests that intracellular growth is not required for the initial stages of intestinal infection, but replication in some as-yet-unidentified cell type in the gut becomes increasingly more important as the infection proceeds. Multicolor flow cytometry can discriminate eight different subsets of myeloid-derived phagocytes that are unique to the gut. Preliminary data provided here verified that intestinal tissue contained at least one cell type that could support intracellular growth of Lm, and ruled out Ly6Chi monocytes and all three subsets of conventional dendritic cells as the intracellular niche. The primary goal of this proposal is to identify the cell type(s) in the gut that support intracellular growth of Lm and to define the innate immune response of intestinal cell types that that interact primarily with either intracellular or extracellular Lm. We hypothesize that Lm initially interact primarily with cell types that that they cannot efficiently invade or survive in, and that later in the course of infection, the bacteria shift to a cell type that serves as a protected intracellular growth niche.!In Aim 1, four candidate intestinal myeloid cell types will be sort purified, infected directly ex vivo and assayed for both intracellular localization and replication. In Aim 2, flow cytometry will be used to identify Lm-associated cells in the lamina propria and submucosa of the ileum and colon as well as the MLN that drain each of these tissues (SI-MLN and LI-MLN) to track the fate of Lm that invade the gut mucosa in mice. In Aim 3, we will define the initial response of all eight subsets of intestinal myeloid cells by measuring the production of cytokines known to be triggered by either host cell surface bound or cytosolic receptors. These exploratory studies will fill a key knowledge gap in the field by defining the early events that occur in the gut during foodborne transmission of Lm. !